The Val-9Ala polymorphism affects the secondary structure of the import sequence in mitochondrial manganese superoxide dismutase (MnSOD). The protein encoded by the Val allele exhibited decreased import in isolated rat liver mitochondria. Furthermore, Val homozygotes had an increased risk of nonfamilial cardiomyopathy in Japanese patients. In contrast, the Ala/Ala genotype was associated with increased risk of breast cancer in premenopausal women. The goals of this project are to develop preliminary results pertaining to the effects of these two MnSODs on mitochondrial and cellular levels of reactive oxygen species (ROS) that would lead to future in depth studies on how the Ala/Ala genotype may contribute to increased breast cancer risk. The Specific Aims of this project are to: 1) Determine the relationship between mitochondrial MnSOD protein levels, MnSOD activity, and superoxide and hydrogen peroxide production. Mitochondria will be isolated from several human breast cell lines with different MnSOD genotypes. The specific activity of MnSOD will be determined by determining enzyme activity and protein levels by Western blotting. The activity of mitochondrial glutathione peroxidase and GSH/GSSL levels will also be determined. 02 consumption and superoxide and hydrogen peroxide production will be determined using chemiluminescent probes during incubation under basal conditions that support mitochondrial respiration and upon treatment to induce oxidative stress. 2) Determine if the mitochondrial import of MnSOD Ala and MnSOD Val differ in mitochondria isolated from human breast cells and, through cross incubations, that the differences are solely due to the MnSOD import sequence differences as opposed to differences in mitochondria protein import capabilities. Mitochondria will be isolated from human breast cell lines, homozygous and heterozygous for the polymorphisms and import rates and levels determined using fusion proteins comprised of the Ala or Val import domains linked to a reporter. The Val and Ala constructs will each be incubated with mitochondria from Val/Val Val/Ala and Ala/Ala cells for comparison. 3) Determine the relationships among MnSOD protein levels, MnSOD activity, and markers of oxidative damage in Ala/Ala, Val/Val, and Val/Ala intact breast cells. Basal and induced levels of mitochondria superoxide, cellular hydrogen peroxide, mitochondrial DNA oxidative damage, protein oxidative damage (i.e. protein carbonyls), and activities of iron-cluster proteins (i.e. aconitase) will be compared and related to MnSOD protein level and activity. 4) From a single breast cell line, develop cell sublines expressing either MnSOD Ala or MnSOD Val to provide a cell culture model for future in depth investigation of potential mechanisms behind the increased breast cancer risk associated with the Ala/Ala genotype. Based on the results of the studies to be conducted in aims 1-3, an appropriate breast cell line(s) will be selected and a gene knock out approach used to develop the sublines referred to. The advantage of this approach is that it will provide two sublines of identical genetic backgrounds and biochemistry except for the differences in the MnSOD import sequences